One of the hurdles facing molecular medicine is the targeted delivery of therapeutic agents such as DNA or RNA molecules. An emerging strategy is the construction of non-viral vectors, such as cationic polymers and cationic lipids, which bind and condense nucleic acids. These non-viral cationic vectors possess many advantages over viral gene vectors, as they are non-immunogenic, non-oncogenic and easy to synthesize [1-4]. Currently, several synthetic polycationic polymers are being developed for nucleic acid delivery. Among these, polyethylenimines (PEIs) are considered promising agents for gene delivery [5].
PEIs are water-soluble, organic macromolecules that are available as both linear and branched structures [6]. PEIs change their degree of ionization over a broad range of pH, since every third atom in their backbone chain is an amino nitrogen, that can be protonated. Approximately 55% of the nitrogens in PEIs are protonated at physiological pH [7]. They possess high cationic charge density, and are therefore capable of forming non-covalent complexes with nucleic acids. Furthermore, their physicochemical and biological properties can be altered by various chemical modifications [8]. PEI-based complexes (also known as polyplexes) can be endocytosed by many cell types [9]. Following internalization of the polyplexes, endosome release and high efficiency gene transfer are driven by the “proton sponge effect” [10]. The ability of PEI to condense DNA appears to be an important factor in delivering large DNA constructs into many cell types.
The major concern in the utilization of PEIs as delivery carriers is toxicity, due to their high positive surface charge, which may lead to non-specific binding [11]. Recent attempts have been made to improve the selectivity and biocompatibility of non-viral vectors. This has led to the modification of PEI molecules with polyethylene glycol (PEG), in order to shield the PEI particle [12]. The conjugation of heterobifunctional PEG groups to PEI facilitates coupling of the PEI to a targeting ligand, which provides efficient gene delivery into cells harboring the cognate receptor [12]. We have previously described the generation of targeting vectors, demonstrating the difference between branched PEI (brPEI-EGF) and linear PEI (LPEI) tethered to EGF as targeting vectors [13, WO 2004/045491, WO 2010/073247]. Current methods of synthesis are unsatisfactory in that they result in insufficiently homogeneous products. There is thus a pressing need for methods that can provide efficient conjugation of targeting moieties to the LPEI-PEG in a reproducible manner to produce homogenous batches of products that can be reliably used in methods for treating cancer.